The advent of DNA microarray technology makes it possible to build an array of hundreds of thousands of DNA sequences in a very small area, such as the size of a microscopic slide. See, e.g., U.S. Pat. Nos. 6 375,903 and 5,143,854, each of which is hereby incorporated by reference in its entirety. The disclosure of U.S. Pat. No. 6,375,903, also incorporated by reference in its entirety, enables the construction of so-called maskless array synthesizer (MAS) instruments in which light is used to direct synthesis of the DNA sequences, the light direction being performed using a digital micromirror device (DMD). Using an MAS instrument, the selection of DNA sequences to be constructed in the microarray is under software control so that individually customized arrays can be built to order. In general, MAS based DNA microarray synthesis technology allows for the parallel synthesis of over 786,000 unique oligonucleotides in a very small area of a standard microscope slide. The microarrays are generally synthesized by using light to direct which oligonucleotides are synthesized at specific locations on an array, these locations being called features. Typically, one nucleotide sequence is synthesized at each feature of the array, i.e., there are multiple probes in each feature, but all those probes have the same nucleotide sequence. For certain applications, oligonucleotides of different sequences can be present within one feature of the array, and the ratio and direction (5′-3′, or 3′-5′) of these oligonucleotides can be controlled.
With the availability of the entire genome of hundreds of organisms, for which a reference sequence has generally been deposited into a public database, microarrays have been used to perform sequence analysis on DNA isolated from such organisms. Microarray methods that for example, allow the measurement of changes in DNA copy number are useful for the determination of chromosomal aberrations in higher eukaryotes that are often linked to disease states. Changes in copy number are typically the result of amplification or deletions of stretches of chromosomes. While large amplification and deletion or translocations can be readily detected by traditional karyotyping methods, the amplification or deletion of smaller DNA fragments within a chromosome can be difficult or impossible to detect by these methods. Accordingly, it has become increasingly important for genetic analysis to utilize the most accurate oligonucleotide probes.
Recently, several research groups have developed methods to optimize probes. For example, to avoid cross-hybridization of highly similar sequences on a microarray, researchers have developed an approach to determine the optimal number and length of gene-specific probes for accurate transcriptional profiling studies. The study surveyed probe lengths from 25 to 1000 nt. It was found that long probes yielded a better signal intensity than short probes. However, the signal intensity of short probes could be improved by addition of spacers or using higher probe concentration for spotting. (see Chou et al., Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression. Nucleic Acids Res. 2004 Jul. 08;32(12):e99.) It is believed that alternative methods for optimizing probes for use in identifying genetic modifications would be a desirable contribution to the art.